The p53 tumor suppressor protein is a key component of the cellular response to stress. It is a homo-tetrameric, sequence-specific transcription factor activated by DNA damage, hypoxia, heat shock, and other types of stress and regulates DNA repair, cell cycle arrest, senescence, metabolism and apoptosis. It is maintained at low levels in unstressed cells but becomes stabilized and activated following DNA damage through extensive post-translational modification (PTM). Our research has focused on identifying and exploring the biological roles of p53 PTMs to better understand how they modulate p53 functions. The tandem N-terminal transactivation domains (TADs) of p53 are crucial for p53's activity as a transcription factor. The two subdomains, TAD1 (residues 1-40) and TAD2 (residues 35-59), interact with several domains of the transcriptional coactivator p300. However, the two subdomains can function independently of one another, suggesting the existence of distinguishing transcriptional cofactors for TAD1 and TAD2 in which the interaction may be differentially regulated by p53 phosphorylation. To identify distinct interacting partners for TAD1 and TAD2, peptides comprising TAD1 (residues 9-33) or TAD2 (residues 35-59), with and without phosphorylation at Thr 18 or Ser 46, respectively, were synthesized and covalently attached to biotin at the N-termini. We used these peptides as a bait for pulldown of interacting proteins from nuclear extracts prepared from MCF7 cells treated with etoposide; reductive dimethylation of peptides followed by mass spectrometry analysis was used to identify and quantitatively compare the interactors to discriminate between those that preferentially interact with TAD1 and TAD2 subdomains. Our preliminary experiments using biological triplicate pulldowns have identified a list of potential interactors that show a preference for either unmodified or modified p53 in untreated cells or following etoposide treatment. In addition to known binding partners of p53 TAD1 and TAD2, we identified several new interactors. The C-terminus of p53 exhibits a diverse array of post-translational modifications, including phosphorylation, methylation, acetylation, ubiquitination, sumoylation, neddylation and hydroxylation that are primarily localized to the terminal thirty residues of the protein. We have shown that p53 can be both mono- and dimethylated on Lys382, with the former modification repressing p53 transcriptional activity and the latter promoting DNA repair, in addition to demonstrated acetylation and ubiquitination of the same site. SETD8 monomethylates p53 on lysine 382, attenuating p53 pro-apoptotic and growth arrest functions. Using a high-content imaging siRNA screen and a chemical screen, in a collaboration with Drs. Veschi and Thiele, we identified SETD8 as a suppressor of p53 activity in neuroblastoma cell lines. Genetic or pharmacological inhibition of SETD8 activity resulted in activation of the p53 wild-type pathway by decreasing p53K382me1. Recently, we demonstrated that inhibition of SETD8 overexpression in colon cancer stem cells results in the activation of p53. To identify inhibitors of SETD8 with a lower IC50 and high tolerability in vivo, we are developing an assay utilizing the Rapid Fire 365 MS System for high-throughput screening. This approach is a viable strategy for discovering novel drugs for tumors that have functionally inactivated wild-type p53. p53 point mutations have been reported to occur in approximately half of all tumors, with marked over-representation of specific hot-spot residues. These mutations leave p53 unable to function as a transcription factor and prevent tumor growth. Moreover, many mutant forms of p53 have novel oncogenic activities due to a gain-of-function mechanism. One characteristic of many of the hot-spot p53 mutants is their structural instability with partial unfolding and the formation of aggregates similar to those seen in amyloid diseases, thus resulting in protein inactivation. Recently, Padmanabhan et al. (Nat Commun. 9(1):1270 (2018)) reported that the deubiquitinase inhibitor, PR-619, specifically targets the hot-spot mutant p53-R175H for degradation. PR-619 disrupts the interaction between p53-R175H and a deubiquitinase, ubiquitin-specific protease 15 (USP15), which leads to ubiquitination and degradation of the mutant protein. Based on these findings, we have chosen to explore other members of the deubiquitinating enzyme (DUB) family as potential modulators of mutant p53 protein stability. Our approach is to conduct CRISPR-interference and CRISPR-activation screens in ovarian cancer cell lines with varying p53 status using DUB-targeting sgRNAs. We hope to identify DUBs that contribute to mutant p53 stability that can be further scrutinized as druggable targets. The ability to selectively target DUBs will be crucial since they take on several roles in the ubiquitin pathway. As the catalytic domains of Ubiquitin-specific proteases (USPs) are highly conserved, regions outside the catalytic domain must be explored for potential drug targeting. The N-terminal domains adjacent to the catalytic domain vary significantly among USPs, making these promising regions for finding unique small molecule-binding pockets. Eventually, sequence and structure data can be used to help develop a rational screen of small molecule and/or peptide mimetic compounds to find more suitable candidates to disrupt the accumulation of mutant p53 and/or rescue p53 wildtype tumor suppressive function. Our work encompasses several different p53 forms including the WT and various hot-spot mutations in a few relevant tumor types. Our future aims are to be able to recognize the functional, metabolic and transcriptional differences these mutations can have in their respective tumors, and the development of molecules, peptides and gene therapy methods to overcome these alterations.